1. Field of the Invention
The present invention pertains in general stress-response proteins and to polynucleotides encoding such factors. The present application pertains in particular to the HypB protein of chlamydia bacteria, specifically to the HypB protein from Chlamydia psittaci and Chlamydia trachomatis, to fragments and polypeptide analogs thereof, and to polynucleotides encoding the same. The HypB protein is a member of a highly conserved family of stress response proteins referred to as HSP60. The HypB protein has a number of acronyms such as the chlamydial GroEL, the chlamydial 57kD antigen and the chlamydial HSP60.
2. Background Information
Members of the genus Chlamydia are obligate intracellular bacteria that are differentiated from all other prokaryotes by their unique intracellular growth cycle. Two species of Chlamydia exist; C. trachomatis, strictly a human pathogen, and C. psittaci, a pathogen of lower mammals. Chlamydiae primarily infect mucosal epithelia, and in humans C. trachomatis causes a formidable group of infections, some of which can progress to severe complications including blindness, infertility, and perhaps arthritis. The most significant of these in the numbers of people afflicted is trachoma, the leading cause of preventable blindness in the world (Jones, Trans. Ophthalmol. Soc.(U.K.), 95, 16(1975)).
Though the pathogenic events that lead to development of severe and often debilitating, post-infection sequelae are not known, an immunological mechanism has been suggested based on studies of human trachoma and sub-human primate models of ocular chlamydial infection (Dawson, Human Chlamydial Infections (1978); Grayston, et al., Rev. Infect. Dis., 7,717 (1985); Silverstein, Invest. Ophthalmol., 13, 560 (1974); Collier, Arch. ges. Virusforsch., 22, 280 (1967)).
Early studies in humans and in sub-human primates indicate that prior vaccination with killed chlamydiae frequently results in more severe trachoma upon reinfection (Wang, et al., Am. J. Ophthalmol., 63, 1615 (1967); See Wang, et al., Am. J. Ophthalmol., 63, 1133 (1967); Grayston, et al., Ann. N.Y. Acad. Sci., 98, 352 (1962); Woolridge, et al., Am. J. Ophthalmol., 63, 1645 (1967); Bell, et al., Am. J. Trop. Med. Hyg. 18, 568 (1969)). Moreover, in some individuals with trachoma, chlamydial antigens and DNA are detected in conjunctival tissue in the absence of cultivatable chlamydia (Wilson, et al., Arch. Ophthalmol., 104, 688 (1986); Schachter, et al., J. Infect. Dis., 158 1347 (1989)). These data support the hypothesis of an immunologically mediated pathogenesis.
C. trachomatis infection of non-human primates and C. psittaci infection of guinea pigs are good model systems for studying chlamydial pathogenesis. Previous studies using these models show that repeated exposure to infectious chlamydiae is necessary to establish the chronic inflammation characteristic of trachoma (Monnickendam, et al., Br. J. Ophthalmol., 64, 284 (1980); Taylor, et al., Invest. Ophthalmol. Vis. Sci., 23, 507 (1982)). Repeated challenge with infectious chlamydiae results in an atypical infection of shortened duration in which chlamydia are difficult to reisolate, and severe ocular disease results; thus suggesting that immune responses are partly protective, but also deleterious. Repeated infection produces a submucosal cellular infiltrate of lymphocytes and macrophages (Patton, et al., J. Infect. Dis., 153, 870 (1986), Monnickendam, supra, and Taylor, supra) like that observed in individuals with trachoma (Hogan & Zimmerman, Ophthalmic Pathology, 240-244 (1962)). Collectively, the human and animal studies argue for a pathogenic role of delayed hypersensitivity (DH).sup.1 in chlamydial disease.
The most direct evidence for DH in pathogenesis of chlamydial disease comes from the observations that a crude extract of viable chlamydiae elicits severe ocular inflammation in immune animals (Watkins, et al., Proc. Natl. Acad. Sci. (USA), 83, 7480 (1986); Taylor, et al., J. Immunol., 138, 3023 (1987)).
In immune guinea pigs, this extract produces an ocular inflammatory response whose histopathology is consistent with human trachoma and chlamydial-induced tubal infertility. Those results support the hypothesis that the host's immune response to chlamydial infection is, in part, deleterious (Moller, et al., Br. J. Vener. Dis. 55, 422 (1979); Hogan, supra, and Watkins, supra).
The inflammation elicited by a chlamydial antigen was clinically and histologically identical to that caused by primary infection which suggested the pathogenesis of the ocular disease was immunologically mediated. This also suggested that the pathogenesis of recurrent chlamydial disease was not due to active infection (Watkins, supra). Identifying hypersensitivity as a major pathogenetic mechanism was important not only in understanding chlamydial associated disease processes but also in establishing future strategies to control chlamydial diseases by immunoprophylaxis. Ocular delayed hypersensitivity has been shown to be induced at mucosal surfaces other than conjunctival-i.e., intestinal or vaginal. Primary chlamydial infection at one mucosal site can elicit a delayed hypersensitivity reaction at either the same or different mucosal surfaces and can contribute to the pathogenesis of chlamydial disease in humans (Watkins, supra).
Recent data suggested that the pathogenesis of guinea pig inclusion conjunctions (GPIC) was mediated by delayed hypersensitivity to an antigen common to strains of both chlamydial species (Watkins, supra).
Prior to the present invention, the biologically active antigen had not been identified. As indicated above, a crude chlamydial extract elicited ocular and dermal delayed hypersensitivity. Lipopolysaccharide (LPS) is a major component of this crude extract and was a suspected antigen. Purified LPS did not elicit hypersensitivity in immune animals but this discovery did not preclude the possibility that the allergen was composed of a complex of LPS with protein, carbohydrate, or lipid (Watkins, supra). Development of a vaccine for trachoma and other chlamydial diseases, which would preclude the deleterious immune response, required identification of the antigen.
The major outer membrane protein (MOMP) is the major structural protein of chlamydiae and is immunogenic (Taylor, et al. J. Immunol., 138, 3023 (1987), supra). Purified MOMP, however, did not elicit an ocular DH response in immune animals. The crude extract of chlamydial elementary bodies (EBs), as indicated above, was biologically active but the exact nature of the extract remained to be determined since neither LPS nor MOMP (components of this extract) elicited an inflammatory response in purified form. Prior to the present invention, the antigenic determinant and inflammatory response elicited by the crude extract was thought to be caused by LPS in conjunction with an alternation in membrane permeability induced by the extraction solution (Taylor, supra). The stimulus for the inflammation response in trachoma has been the subject of ongoing speculation.
The Applicants have isolated and purified a chlamydial antigenic protein responsible for the ocular delayed hypersensitivity inflammatory response from Chlamydia psittaci and Chlamydia trachomatis. The immunologically bioactive component is the HypB protein of the present invention (Morrison, et al., J. Exp. Med., 169, 663 (1989)). Also described is a HypA antigenic protein (Morrison, et al., supra). The chlamydial gene of the C. psittaci and C. trachomatis that encodes the HypB protein have been cloned, and the recombinantly produced protein of C. psittaci has been shown to elicit an ocular DH response in immune guinea pigs. The sequencing of the gene revealed a close relatedness to the heat-shock or stress proteins GroEL of Escherichia coli, HtpB of Coxiella burnetii, 65 k of Mycobacterium tuberculosis, and Hsp60 of Saccharomyces cerevisiae.
While several antigenic detection diagnostic tests for chlamydia are available, none use immunological reagents specific for the HypB protein of the present invention (Chernesky, et al., J. Infect. Dis. 154, 141 (1986); Howard, et al., J. Clin. Microbiol., 23, 329 (1986); Tam, et al., N. Engl. J. Med., 310, 1146 (1984)). The present invention allows for the preparation of peptides analogous to chlamydial-specific determinants for use in monoclonal antibody and monospecific-polyclonal antisera preparation which can be used to identify chlamydiae in clinical specimens or cell culture of chlamydial isolates. Synthetic peptides analogous to the chlamydial-specific determinants can be used in serological assays to diagnose chlamydial infections. The HypB chlamydial protein elicits a cell-mediated immune response in addition to an antibody response. Therefore, the present invention allows for the use of a chlamydial-specific region of the protein as a skin test antigen to diagnose or monitor chlamdial infection; analogous to the use of the TB skin test antigen PPD.